Sucrose-gradient fractionation was performed as described earlier28 (link). Briefly, 10–40% sucrose gradients were formed by layering 400 μl of lysis buffer containing 10, 20, 30, or 40% sucrose in a sorvall ultracentrifuge tubes. One milligram of cell extract or 400 μg of molecular weight markers (Sigma MW-GF-1000) was loaded on top of the gradient and ultracentrifuged at 37,000 r.p.m. for 17 h at 4 °C using Sorvall WXUltra100 (Thermo Scientific) in an AH650 rotor. Forty fractions of 45 μl each were collected from the top, and alternate fractions were electrophoresed on 7.5% SDS-PAGE and analyzed by immunoblotting with desired antibodies.
Sorvall wx ultra 100
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sorvall WX Ultra 100 is a high-speed centrifuge designed for a wide range of laboratory applications. It is capable of reaching speeds up to 100,000 rpm and can generate centrifugal forces up to 802,000 x g. The centrifuge features a compact design and user-friendly interface.
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Lab products found in correlation
Mwgf1000, by Merck Group (1 mentions)
Mnase, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Cycloheximide (chx), by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Icap 6500 duo, by Thermo Fisher Scientific (1 mentions)
Axs d8 advance powder x ray diffractometer, by Bruker (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Diic18 3, by Thermo Fisher Scientific (1 mentions)
Zetaview, by Particle Metrix (1 mentions)
Sorvall evolution rc, by Thermo Fisher Scientific (1 mentions)
Sorvall rc 6 plus, by Thermo Fisher Scientific (1 mentions)
Uc tubes, by Beckman Coulter (1 mentions)
Mircury exosome serum plasma kit, by Qiagen (1 mentions)
9 protocols using sorvall wx ultra 100
1
Sucrose Gradient Fractionation for Protein Analysis
2
Preparation of Mononucleosomes from HeLa Cells
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Nucleosomes were prepared freshly from HeLa cells as described previously45 (link). Briefly, the nuclear pellet from HeLa cells was digested with MNase (0.2 Units/µl; Sigma) and extracted with TE buffer for 1 h. The mononucleosome was separated via sucrose gradient (5–40%) ultracentrifugation using Sorvall WXUltra100 (Thermo Fischer Scientific) with AH650 rotor for 16 h at 207,203 × g. For further analysis, these fractions were pooled and concentrated.
3
Polysome Fractionation and RNA Analysis
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Total polysome was isolated and fractionated using sucrose gradient fractionation as described elsewhere [43 (link)]. Briefly, cells were incubated with 100 μg/ml of cycloheximide (Sigma) at 37 °C for 10 min. The cells were lysed in polysome extraction buffer (10 mM HEPES pH 8.0, 5 mM MgCl2, 25 mM KCl, 1% Triton X100, 1 mM DTT, 1% sodium deoxycholate, 5 mM vanadyl ribonucleoside complex, 1X protease inhibitor cocktail (Roche) containing 100 μg/ml cycloheximide). The cytoplasmic extract was isolated, and total RNA was quantitated using NanoDrop. An equal amount of cytoplasmic lysate was fractionated on a sucrose gradient (10–50%) and ultracentrifuged at 190,000 g for 90 min at 4 °C in Sorvall WXUltra100 (Thermo Fischer Scientific) with AH650 rotor. Total RNA was isolated and analyzed from each volume 500 μl fraction and percent of RNA was calculated.
4
Bioaccessibility Measurement of Curcumin
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After the small intestinal phase, the samples were separated into two portions: a micelle sample and a total digest sample. To isolate the mixed micelle fraction containing solubilized curcumin, a 20 mL aliquot of the entire digesta was centrifuged (38,200 g and 20°C for 30 min) using a T-865 rotor (Sorvall WX Ultra 100; Thermo Scientific, Asheville, NC, United States) and the clear supernatant was collected. To separate hydrophobic curcumin, the mixed micelle fraction and the whole digest fraction were dispersed in chloroform, vortexed and centrifuged for 60 min at 3,800 g. The following equation was used to calculate the bioaccessibility of curcumin:
In this formula, the measured curcumin concentration in the micelle phase is represented by Cmicelle, and the actual curcumin concentration in the intestinal digesta is represented by Cdigesta.
In this formula, the measured curcumin concentration in the micelle phase is represented by Cmicelle, and the actual curcumin concentration in the intestinal digesta is represented by Cdigesta.
5
Physicochemical Characterization of NiPB@AgNC
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The as-synthesized nanocomposite was thoroughly characterized by several physicochemical techniques. Initially, 50 ml of NiPB@AgNC (obtained after second step of reaction) was centrifuged for 40 min at 17,700 r.p.m. at 15°C using ultracentrifuge (Thermo Fisher Scientific, Sorvall-WX ultra 100). The intense loose ash yellow-colored pellet obtained after centrifugation was used for further characterizations and cell viability studies and antibacterial studies. The absorbance, crystallinity and purity of NiPB@AgNC were monitored by UV–Visible spectroscopy (JASCO dual-beam spectrophotometer [Model V-570]) and x-ray diffraction (XRD) analysis using Bruker AXS D8 Advance Powder x-ray diffractometer (CuKαλ = 1.5406 Å radiation), respectively. The size, morphology and surface charge (zeta potential) were determined by transmission electron microscopy (TEM: FEI Tecnai F12, Philips Electron Optics, Eindhoven, Holland) and DLS, respectively. ICP-OES [(iCAP-6500 DUO), Thermo Fisher Scientific, Cambridge, UK] was used for the determination of silver, nickel and iron in the loose pellet.
6
Isolation of Cell Line-Derived Exosomes
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ALL cell lines were cultured in serum-free DMEM/F12 (Ham) under standard conditions (37 °C, 5% CO2, 95% humidity) for 48 h prior to isolation of ALL cell line-derived exosomes. Supernatant was cleared by differential centrifugation (200× g for 10 min, 300× g for 10 min, 1500× g for 20 min, 2500× g for 20 min, 4500× g for 30 min, 14,000× g for 30 min). Exosomes were pelleted down by ultracentrifugation (Sorvall WX Ultra 100 equipped with Sorvall SureSpin 630, Thermo Fisher Scientific, Dreieich, Germany) for 150 min at 100,000× g. Exosomes were resuspended in PBS (Thermo Fisher, Dreieich, Germany), filter-sterilized (0.22 μm, Sartorius, Göttingen, Germany) and stored at −80 °C. Where indicated, exosomes were stained with DiIC18(3) (Thermo Fisher, Dreieich, Germany) according to manufacturer’s instructions.
7
Isolation and Characterization of Extracellular Vesicles
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EV were isolated by differential centrifugation. HCT116 and DLD-1 cells were cultured in McCoy’s 5A medium supplemented with 10% exosome-depleted fetal calf serum. Briefly, cancer cell line culture medium was centrifuged at 300 g for 10 min to pellet cells, supernatant was transferred to new falcon tubes and then centrifuged at 2000 g for 20 min to pellet dead cells and apoptotic bodies (Rotanta 460RC, Hettich). Supernatant was transferred to the high-speed centrifugation tubes and large EV were removed by ultracentrifugation at 13,500 g for 30 min (Sorvall Evolution RC, Thermo Fisher Scientific). After filtration of the supernatant through a 0.22 µm syringe filter, small EV were pelleted by ultracentrifugation at 246,836 g for 90 min (rotor T-1250), suspended in ice-cold PBS and collected after another ultracentrifugation at 92,500 g for 90 min (rotor type 25, Sorvall WX Ultra 100, Thermo Fisher Scientific). The EV pellet was resuspended in cold PBS for further use. EV count was determined using a Nanoparticle tracking analyzer (ZetaView, Particlemetrix). Results were analyzed with ZetaView software. EV were added to macrophages at a concentration of 2×109/1×106 cells. We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV200065).37 (link)
8
Synthesis of Au-C and Au-P Nanoparticles
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HAuCl4 (200 μL, 10−2 M) solution was added to a 4.8 mL water fraction of Curcumin and Paclitaxel (10 μg/mL). The total volume of the reaction mixture was adjusted to 5 mL in all experiments. The resulting Au-C and Au-P were purified by ultracentrifugation at 15,000 rpm (25,000× g) for 40 min at 15 °C (Sorvall WX ultra-100, Thermo scientific, Benguluru, India) [15 (link),18 (link)].
9
Extracellular Vesicle Isolation Protocols
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EVs were isolated from plasma using differential ultracentrifugation (UC) as described previously [27 (link)] or the miRCURY Exosome Serum/Plasma Kit (Qiagen, Hilden, Germany).
In the case of UC, 500 μL plasma samples were transferred into 15 mL sterile high-speed centrifuge tubes (VWR-Avantor, Philadelphia, PA, USA), filled up with sterile 1× phosphate-buffered saline (PBS) and centrifuged twice at 10,000× g for 30 min at 4 °C in a Sorvall RC 6 Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Supernatants were then transferred into UC tubes (Beckman Coulter, Brea, CA, USA), equilibrated with sterile 1× PBS, and spun twice at 70,000× g for 1 h at 4 °C in the Sorvall WX Ultra 100 centrifuge (Thermo Fisher Scientific). The EV enriched pellets were resuspended in 100 μL sterile PBS and stored at −80 °C until used. EV enrichment with the miRCURY Kit was performed as described [25 (link)]. Debris was cleared from 500 μL plasma samples with thrombin and subsequent centrifugation at 10,000× g for 5 min at RT. Samples were then incubated with Precipitation Buffer overnight at 4 °C and centrifuged twice (500× g, 5 min at RT). Supernatants were discarded, EV enriched pellets were resuspended in 270 μL of Resuspension Buffer and stored at −80 °C until used.
In the case of UC, 500 μL plasma samples were transferred into 15 mL sterile high-speed centrifuge tubes (VWR-Avantor, Philadelphia, PA, USA), filled up with sterile 1× phosphate-buffered saline (PBS) and centrifuged twice at 10,000× g for 30 min at 4 °C in a Sorvall RC 6 Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Supernatants were then transferred into UC tubes (Beckman Coulter, Brea, CA, USA), equilibrated with sterile 1× PBS, and spun twice at 70,000× g for 1 h at 4 °C in the Sorvall WX Ultra 100 centrifuge (Thermo Fisher Scientific). The EV enriched pellets were resuspended in 100 μL sterile PBS and stored at −80 °C until used. EV enrichment with the miRCURY Kit was performed as described [25 (link)]. Debris was cleared from 500 μL plasma samples with thrombin and subsequent centrifugation at 10,000× g for 5 min at RT. Samples were then incubated with Precipitation Buffer overnight at 4 °C and centrifuged twice (500× g, 5 min at RT). Supernatants were discarded, EV enriched pellets were resuspended in 270 μL of Resuspension Buffer and stored at −80 °C until used.
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